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1.  Caerulomycin A Suppresses Immunity by Inhibiting T Cell Activity 
PLoS ONE  2014;9(10):e107051.
Background
Caerulomycin A (CaeA) is a known antifungal and antibiotic agent. Further, CaeA is reported to induce the expansion of regulatory T cell and prolongs the survival of skin allografts in mouse model of transplantation. In the current study, CaeA was purified and characterized from a novel species of actinomycetes, Actinoalloteichus spitiensis. The CaeA was identified for its novel immunosuppressive property by inhibiting in vitro and in vivo function of T cells.
Methods
Isolation, purification and characterization of CaeA were performed using High Performance Flash Chromatography (HPFC), NMR and mass spectrometry techniques. In vitro and in vivo T cell studies were conducted in mice using flowcytometry, ELISA and thymidine-[methyl-3H] incorporation.
Results
CaeA significantly suppressed T cell activation and IFN-γ secretion. Further, it inhibited the T cells function at G1 phase of cell cycle. No apoptosis was noticed by CaeA at a concentration responsible for inducing T cell retardation. Furthermore, the change in the function of B cells but not macrophages was observed. The CaeA as well exhibited substantial inhibitory activity in vivo.
Conclusion
This study describes for the first time novel in vitro and in vivo immunosuppressive function of CaeA on T cells and B cells. CaeA has enough potential to act as a future immunosuppressive drug.
doi:10.1371/journal.pone.0107051
PMCID: PMC4186789  PMID: 25286329
2.  Lipidated promiscuous peptide augments the expression of MHC-II molecules on dendritic cells and activates T cells 
Background & objectives:
In spite of the fact that BCG is the most widely used vaccine, tuberculosis (TB) continues to be a major killer disease in TB-endemic regions. Recently, many emerging evidences from the published literature indicate the role of environmental mycobacteria in blocking the processing and presentation of BCG antigens and thereby impairing with suboptimal generation of protective T cells. To surmount this problem associated with BCG, we constructed a novel lipopeptide (L91) by conjugating a promiscuous peptide consisting of CD4+ T-helper epitope of sequence of 91-110 of 16 kDa antigen of Mycobacterium tuberculosis to Pam2Cys, an agonist of Toll-like receptor-2.
Methods:
Mice were immunized subcutaneously with 20 nmol of L91, followed by a booster with 10 nmol, after an interval of 21 days of primary immunization. Animals were sacrificed after seven days of post-booster immunization. L91 induced immune response was characterized by the expression of MHC-II and CD74 on the surface of dendritic cells (DCs) by flowcytometry. Cytokines (IL-4, IL-10, IFN-γ) secretion and anti-peptide antibodies were measured by ELISA.
Results:
Self-adjuvanting lipopeptide vaccine (L91) was directly bound to MHC-II molecules and without requiring extensive processing for its presentation to T cells. It stimulated and activated dendritic cells and augmented the expression of MHC-II molecules. Further, it activated effector CD4 T cells to mainly secrete interferon (IFN)-γ but not interleukin (IL)-4 and IL-10. L91 did not elicit anti-peptide antibodies.
Interpretation & conclusions:
The findings suggest that L91 evokes maturation and upregulation of MHC class II molecules and promotes better antigen presentation and, therefore, optimum activation of T cells. L91 mainly induces effector Th1 cells, as evidenced by predominant release of IFN-γ, consequently can mount favourable immune response against M. tuberculosis. As L91 does not provoke the generation of anti-peptide antibodies, there is no fear of the efficacy of the vaccine being neutralized by pre-existing anti-mycobacterial antibodies in TB-endemic population. In conclusion, L91 may be considered as a future potential candidate vaccine against TB.
PMCID: PMC3928704  PMID: 24434326
BCG; L91; Pam2Cys; promiscuous peptides; Th1 – tuberculosis
3.  Genome sequencing, annotation and comparative genomic analysis of Shigella dysenteriae strain SD1D 
Gut Pathogens  2014;6:28.
Background
Shigellosis is an acute form of gastroenteritis caused by the bacteria belonging to the genus Shigella. It is the most common cause of morbidity and mortality in children. Shigella belongs to the family Enterobactericeae, which is a Gram-negative and rod shaped bacterium. In the present study, we report the draft genome of Shigella dysenteriae strain SD1D, which was isolated from the stool sample of a healthy individual.
Results
Based on 16S rRNA gene sequence and phylogenetic analysis, the strain SD1D was identified as Shigella dysenteriae. The draft genome of SD1D consisted of 45, 93, 159 bp with a G + C content of 50.7%, 4, 960 predicted CDSs, 75 tRNAs and 2 rRNAs. The final assembly contained 146 contigs of total length 45, 93, 159 bp with N50 contig length of 77, 053 bp; the largest contig assembled measured 3, 85, 550 bp.
Conclusions
We have for the first time performed the whole genome sequencing of Shigella dysenteriae strain SD1D. The comparative genomic analysis revealed several genes responsible for the pathogenesis, virulence, defense, resistance to antibiotics and toxic compounds, multidrug resistance efflux pumps and other genomic features of the bacterium.
doi:10.1186/1757-4749-6-28
PMCID: PMC4099087  PMID: 25028600
Shigella dysenteriae; Shigellosis; G + C content; EzTaxon; RAST
4.  Genome sequencing, annotation and analysis of Salmonella enterica sub species salamae strain DMA-1 
Gut Pathogens  2014;6:8.
Background
The genus Salmonella is Gram-negative which belongs to the family Enterobacteriaceae. In this study, we have sequenced the whole genome of the strain DMA-1, which was isolated from mouse stool sample and identified as Salmonella enterica subspecies salamae.
Results
The strain DMA-1 was closely related at the 16S rRNA gene sequence level with the members of the genus Salmonella: Salmonella enterica subspecies salamae DSM 9220T (100%), followed by Salmonella enterica subspecies diarizonae (99.1%), Salmonella enterica subspecies enterica (99.0%) and Salmonella enterica subspecies indica (98.5%). We obtained the draft genome of S. enterica subspecies salamae strain DMA-1 with a size of 4,826,209 bp and mean G+C content of 52.0 mol%.
Conclusions
We for the first time, sequenced the entire genome of the strain DMA-1 which was isolated from the mouse stool sample and identified it as Salmonella enterica, sub species salamae. Further, we subjected the whole genome sequencing data for annotation that revealed several genes responsible for the pathogenesis, virulence, defense, metabolism and other genomic features.
doi:10.1186/1757-4749-6-8
PMCID: PMC4108123  PMID: 24721679
Salmonella enterica subspecies salamae; RAST; EzTaxon
5.  Correction: Combinatorial Signaling through TLR-2 and CD86 Augments Activation and Differentiation of Resting B Cells 
PLoS ONE  2014;9(1):10.1371/annotation/f0ad0323-84d6-4c21-be15-d07aed1993a1.
doi:10.1371/annotation/f0ad0323-84d6-4c21-be15-d07aed1993a1
PMCID: PMC3888144
6.  Combinatorial Signaling through TLR-2 and CD86 Augments Activation and Differentiation of Resting B Cells 
PLoS ONE  2013;8(1):e54392.
B cells are an integral component in mounting humoral immune responses and they are also crucial in programming T cell mediated immunity. Usually, B cell activation is initiated by recognition of antigen through B cell receptor (BCR), followed by its processing and presentation to T cells. But very little is known about BCR independent activation of B cells. Now, there is an increasing body of evidence indicating the combinatorial effect of innate and adaptive immune components in modulating the functions of B cells. In this study, we demonstrate the activation of resting B cells (RB) by simultaneous involvement of Toll like Receptor-2 (TLR-2) and costimulatory molecule, CD86. Interestingly, these B cells exhibited significant level of activation and proliferation. Furthermore, this process of activation leads to the differentiation of RB cells, preferably into marginal zone precursors (CD19+IgDhiIgMhiCD21/35hiCD23hi) in a shorter time window and showed increased secretion of IgG isotype. These RB cells also showed enhanced antigen uptake capacity. These observations were also substantiated by microarray gene expression results, which strengthen the notion that combinatorial signaling through innate and adaptive immune components enhances B cell mediated immune response. Thus, the present study elucidates a novel BCR independent B cell activation mechanism that links TLR-2 and CD86. Hence signaling of TLRs in conjunction with costimulatory molecules will substantially help in bolstering humoral immune response, which can be extrapolated to formulate vaccination strategies for diseases involving B cell-mediated immunity.
doi:10.1371/journal.pone.0054392
PMCID: PMC3554778  PMID: 23365665
7.  Manipulation of Costimulatory Molecules by Intracellular Pathogens: Veni, Vidi, Vici!! 
PLoS Pathogens  2012;8(6):e1002676.
Some of the most successful pathogens of human, such as Mycobacterium tuberculosis (Mtb), HIV, and Leishmania donovani not only establish chronic infections but also remain a grave global threat. These pathogens have developed innovative strategies to evade immune responses such as antigenic shift and drift, interference with antigen processing/presentation, subversion of phagocytosis, induction of immune regulatory pathways, and manipulation of the costimulatory molecules. Costimulatory molecules expressed on the surface of various cells play a decisive role in the initiation and sustenance of immunity. Exploitation of the “code of conduct” of costimulation pathways provides evolutionary incentive to the pathogens and thereby abates the functioning of the immune system. Here we review how Mtb, HIV, Leishmania sp., and other pathogens manipulate costimulatory molecules to establish chronic infection. Impairment by pathogens in the signaling events delivered by costimulatory molecules may be responsible for defective T-cell responses; consequently organisms grow unhindered in the host cells. This review summarizes the convergent devices that pathogens employ to tune and tame the immune system using costimulatory molecules. Studying host-pathogen interaction in context with costimulatory signals may unveil the molecular mechanism that will help in understanding the survival/death of the pathogens. We emphasize that the very same pathways can potentially be exploited to develop immunotherapeutic strategies to eliminate intracellular pathogens.
doi:10.1371/journal.ppat.1002676
PMCID: PMC3375268  PMID: 22719245
8.  Potential T cell epitopes of Mycobacterium tuberculosis that can instigate molecular mimicry against host: implications in autoimmune pathogenesis 
BMC Immunology  2012;13:13.
Background
Molecular mimicry between microbial antigens and host-proteins is one of the etiological enigmas for the occurrence of autoimmune diseases. T cells that recognize cross-reactive epitopes may trigger autoimmune reactions. Intriguingly, autoimmune diseases have been reported to be prevalent in tuberculosis endemic populations. Further, association of Mycobacterium tuberculosis (M. tuberculosis) has been implicated in different autoimmune diseases, including rheumatoid arthritis and multiple sclerosis. Although, in silico analyses have identified a number of M. tuberculosis specific vaccine candidates, the analysis on prospective cross-reactive epitopes, that may elicit autoimmune response, has not been yet attempted. Here, we have employed bioinformatics tools to determine T cell epitopes of homologous antigenic regions between M. tuberculosis and human proteomes.
Results
Employing bioinformatics tools, we have identified potentially cross-reactive T cell epitopes restricted to predominant class I and II alleles of human leukocyte antigens (HLA). These are similar to peptides of mycobacterial proteins and considerable numbers of them are promiscuous. Some of the identified antigens corroborated with established autoimmune diseases linked with mycobacterial infection.
Conclusions
The present study reveals many target proteins and their putative T cell epitopes that might have significant application in understanding the molecular basis of possible T cell autoimmune reactions during M. tuberculosis infections.
doi:10.1186/1471-2172-13-13
PMCID: PMC3359254  PMID: 22435930
9.  CD40 Signaling Synergizes with TLR-2 in the BCR Independent Activation of Resting B Cells 
PLoS ONE  2011;6(6):e20651.
Conventionally, signaling through BCR initiates sequence of events necessary for activation and differentiation of B cells. We report an alternative approach, independent of BCR, for stimulating resting B (RB) cells, by involving TLR-2 and CD40 - molecules crucial for innate and adaptive immunity. CD40 triggering of TLR-2 stimulated RB cells significantly augments their activation, proliferation and differentiation. It also substantially ameliorates the calcium flux, antigen uptake capacity and ability of B cells to activate T cells. The survival of RB cells was improved and it increases the number of cells expressing activation induced deaminase (AID), signifying class switch recombination (CSR). Further, we also observed increased activation rate and decreased threshold period required for optimum stimulation of RB cells. These results corroborate well with microarray gene expression data. This study provides novel insights into coordination between the molecules of innate and adaptive immunity in activating B cells, in a BCR independent manner. This strategy can be exploited to design vaccines to bolster B cell activation and antigen presenting efficiency, leading to faster and better immune response.
doi:10.1371/journal.pone.0020651
PMCID: PMC3107243  PMID: 21674065
10.  Co-Administration of IL-1+IL-6+TNF-α with Mycobacterium tuberculosis Infected Macrophages Vaccine Induces Better Protective T Cell Memory than BCG 
PLoS ONE  2011;6(1):e16097.
BCG has been administered globally for more than 75 years, yet tuberculosis (TB) continues to kill more than 2 million people annually. Further, BCG protects childhood TB but is quite inefficient in adults. This indicates that BCG fails to induce long-term protection. Hence there is a need to explore alternative vaccination strategies that can stimulate enduring T cell memory response. Dendritic cell based vaccination has attained extensive popularity following their success in various malignancies. In our previous study, we have established a novel and unique vaccination strategy against Mycobacterium tuberculosis (M. tb) and Salmonella typhimurium by utilizing infected macrophages (IM). In short-term experiments (30 days), substantial degree of protection was observed. However, remarkable difference was not observed in long-term studies (240 days) due to failure of the vaccine to generate long-lasting memory T cells. Hence, in the present study we employed T cell memory augmenting cytokines IL-1+IL-6+TNF-α and IL-7+IL-15 for the induction of the enhancement of long-term protection by the vaccine. We co-administered the M. tb infected macrophages vaccine with IL-1+IL-6+TNF-α (IM-1.6.α) and IL-7+IL-15 (IM-7.15). The mice were then rested for a reasonably large period (240 days) to study the bona fide T cell memory response before exposing them to aerosolized M. tb. IM-1.6.α but not IM-7.15 significantly improved memory T cell response against M. tb, as evidenced by recall responses of memory T cells, expansion of both central as well as effector memory CD4 and CD8 T cell pools, elicitation of mainly Th1 memory response, reduction in the mycobacterial load and alleviated lung pathology. Importantly, the protection induced by IM-1.6.α was significantly better than BCG. Thus, this study demonstrates that not only antigen-pulsed DCs can be successfully employed as vaccines against cancer and infectious diseases but also macrophages infected with M. tb can be utilized with great efficacy especially in protection against TB.
doi:10.1371/journal.pone.0016097
PMCID: PMC3023717  PMID: 21283805
11.  CD4+ T-cell memory: generation and multi-faceted roles for CD4+ T cells in protective immunity to influenza 
Immunological reviews  2006;211:8-22.
Summary
We have outlined the carefully orchestrated process of CD4+ T-cell differentiation from naïve to effector and from effector to memory cells with a focus on how these processes can be studied in vivo in responses to pathogen infection. We emphasize that the regulatory factors that determine the quality and quantity of the effector and memory cells generated include (i) the antigen dose during the initial T-cell interaction with antigen-presenting cells; (ii) the dose and duration of repeated interactions; and (iii) the milieu of inflammatory and growth cytokines that responding CD4+ T cells encounter. We suggest that heterogeneity in these regulatory factors leads to the generation of a spectrum of effectors with different functional attributes. Furthermore, we suggest that it is the presence of effectors at different stages along a pathway of progressive linear differentiation that leads to a related spectrum of memory cells. Our studies particularly highlight the multi-faceted roles of CD4+ effector and memory T cells in protective responses to influenza infection and support the concept that efficient priming of CD4+ T cells that react to shared influenza proteins could contribute greatly to vaccine strategies for influenza.
doi:10.1111/j.0105-2896.2006.00388.x
PMCID: PMC2266984  PMID: 16824113
12.  Regulatory role of pro-Th1 and pro-Th2 cytokines in modulating the activity of Th1 and Th2 cells when B cell and macrophages are used as antigen presenting cells 
BMC Immunology  2006;7:17.
Background
Presence of antigen presenting cells, expression of costimulatory molecules, the strength of first signal and cytokine milieu are quite important in influencing the reactivation of differentiated Th1 and Th2 cells.
Results
In the present study, we have analyzed the concerted action of pro-Th1 and pro-Th2 cytokines in the presence of B cells, peritoneal and splenic macrophages as antigen presenting cells and varied concentration of first (anti-CD3 Ab) and second (B7-1 transfectant) signals on the proliferation and cytokine secretion by Th1 and Th2 cells. Interesting observations were made that IFN-γ significantly augmented the secretion of IL-4 by Th2 cells when either B cells or splenic or peritoneal macrophages were used as APC. Further, IFN-γ significantly inhibited the proliferation of Th1 cells only in the presence of peritoneal macrophages. We have also observed that B cells could significantly respond to cytokines to further enhance the proliferation and cytokine release by Th1 and Th2 cells. But not much effect on addition of exogenous cytokines IL-1, IL-4, IL-5, IL-12 was observed on the proliferation of Th1 and Th2 cells in the presence of macrophages. In contrast, both IFN-γ and IL-2 significantly enhanced the production of IL-4 and IL-5 respectively, by Th2 cells in presence of B cells, splenic and peritoneal macrophages. Another important observation was that the addition of B7-1 transfectants in the cultures, which were stimulated with low dose of anti-CD3 Ab significantly, enhanced the proliferation and cytokine secretion.
Conclusion
This study indicates involvement of different type of APCs, cytokine milieu, dose of first and second signals in a concerted manner in the outcome of the immune response. The significance of this study is that the immunization with antigen along with costimulatory molecules may significantly reduce the dose of antigen and can generate better immune response than antigen alone.
doi:10.1186/1471-2172-7-17
PMCID: PMC1550426  PMID: 16889674

Results 1-12 (12)